Sesquiterpene synthases catalyze the cyclization of the universal acyclic precursor, farnesyl diphosphate (1) to any of 200 distinct cyclic sesquiterpene hydrocarbons and alcohols, which in turn can serve as the metabolic precursors of the many thousands of known sesquiterpenoids. These metabolites can display a wide range of antibiotic or other physiological activities, be it antiviral, antibacterial, antifungal, insecticidal, antifeedant, cytostatic, immunosuppressive, or neurotoxic. Extensive investigations with both stereospecifically labeled substrates as well as with various competitive inhibitors, carried out in our own and other laboratories, have led to a general mechanistic and stereochemical model of terpenoid cyclization. We propose to continue and extend ongoing studies of the mechanistic enzymology and molecular genetics of terpenoid cyclizations. The focus will be on a family of microbial sesquiterpene synthases which catalyze the conversion of farnesyl diphosphate (1) to trichodiene (2), aristolochene (3) beta-trans-bergamotene (4), and epi-cubenol (5). In collaboration with Professor Rodney Croteau of Washington State University we will also study a group of monoterpene synthases, including limonene (6) synthase. In order to test and to extend further a general stereochemical model of terpenoid cyclizations, three broad and closely interrelated questions must be addressed: 1) How does a cyclase impose a specific folding pattern or conformation on the substrate FPP and derived intermediates? 2) How does a cyclase manage charged intermediates, including.catalysis of the initial ionization of substrate, through stabilization of cationic intermediates, to termination of the reaction by quenching of positive charge? A related issue is how does the cyclase avoid annihilation which would result from reaction with the highly electrophilic species it must accommodate? 3) What is the nature of the active site? To answer these questions, we have devised a set of mutually complementary experimental approaches, involving: A. Synthesis and cyclization of stereospecifically labeled substrates and intermediates and analysis of the distribution of label in the derived cyclization products by 2H and 13C NMR; B . Purification of sequiterpene cyclases and cloning, sequencing, and overexpression of the relevant structural genes; C. Design, synthesis, and testing of substrate and intermediate analogs as either competitive inhibitors of the normal cyclization or active-site directed and mechanism-based irreversible inactivators of sesquiterpene or monoterpene synthases; D. Isolation and identification of normally enzyme- bound intermediates through rapid quench experiments or the use of anomalous substrates which can only undergo partial reactions. The techniques and experimental approaches developed in the proposed research are expected to be broadly applicable to the understanding of not only terpenoid cyclizations but enzyme mechanistic and biosynthetic investigations in general, as well as the evolution of natural products biosynthetic pathways.